Process for the production of alcoholic beverages using maltseed

ABSTRACT

The invention relates to a process for the production of alcoholic beverages such as beer or whiskey with a mixture of enzymes comprising an endo-β(1,4)-xylanase, an arabinofuranosidase, an alpha-amylase, an endo-protease and a β-( 1,3; 1,4 )-glucanase, and optionally, a saccharifying amylase and/or an exopeptidase. Preferable are mixtures in which the enzymes which are necessary in the brewing process are provided by transgenic seeds. Only a minor amount of malt may be necessary for flavor and color.

This application is a continuation of application No. 09/230,590 filedon Apr. 28, 1999 now U.S. Pat. No. 6,361,808, which is InternationalApplication PCT/EP97/04016 filed on Jul. 23, 1997, which designated theU.S., was published in English, claims the benefit thereof andincorporates the same by reference.

FIELD OF THE INVENTION

The present invention relates to a process for the production ofalcoholic beverages, especially beer and whiskey.

BACKGROUND OF THE INVENTION

Alcoholic beverages such as beer can be manufactured from malted and/orunmalted barley grains. Malt, in addition to yeast, contributes toflavour and colour of the beer. Furthermore, malt functions as a sourceof fermentable sugar and enzymes. Whether malt is used in the brewingprocess depends on the type of beer and on the country where the beer isproduced. In African countries, for example, there is no tradition ofusing malt.

The general process of malting and brewing is recently described by R.C. Hoseney (Cereal Foods World, 39(9), 675-679, 1994). Malting is theprocess of controlled germination followed by controlled drying of thebarley grain. Grain is converted into malt by successive steps ofsteeping, germination, growth and drying (kilning). In this respect, thegermination step is important to obtain expression of a series ofenzymes which enables modification of the endosperm. This modificationproduces fermentable carbohydrates.

The subsequent drying/heating step of the malting process producesflavour and colour due to non-enzymatic browning (Maillard) reactions.

The process of malting is a very complicated and costly part of the beerproduction process. Several disadvantages of the malting process can bementioned:

the enzyme level of malt is variable which leads to unpredictableresults,

not every enzyme activity which is desirable is formed or is formed insufficient amounts during germination, which makes enzymesupplementation necessary,

conditions which favour high flavour and colour may be deleterious forenzyme activity of the malt,

the process is expensive,

10-20% loss in weight occurs during germination, due to respiration andgrowth of rootlets (which are removed during cleaning of the malt),

it is not possible to produce malt at any place which is desirable,because of unfavourable climate conditions,

the use of malt can lead to colloidal instability because ofsolubilization of protein by protease present in the malt,

formation of biogenic amines can occur (J. Food Science 59(5),1104-1107, 1994), which may lead to e.g. histaminic intoxication.

Traditionally, malt was the only source of fermentable carbohydrates andenzymes, and in many countries it still is. However, to date more andmore beers are produced using other sources of carbohydrates than maltand/or barley, i.e. virtually any starch source or liquefied/degradedstarch, the so-called adjuncts. Since malt not only functions as asource of fermentable carbohydrate, but also as a source of enzymes,alternative enzyme sources have to be provided upon substitution of morethan approximately 50% malt with unmalted barley and/or with adjuncts.Moreover, malt gives the beer flavour and colour.

In the production of malt there is a trade-off between flavour andcolour and enzyme activity. A malt providing high flavour and darkcolour can only be produced after more extensive drying at relativelyhigh temperatures. These are conditions which are deleterious for theactivity of an enzyme. Thus the supplementation of enzymes from anexogenous source is necessary from several points of view. In thatrespect, the use of microbial enzymes has been common practice for sometime. For example, for brewing beer grains and/or malted grains areliquefied and saccharified in order to yield fermentable sugars.Liquefaction steps may be improved by the use of thermostablealpha-amylases as described in for instance U.S. Pat. Nos. 4,285,975 or5,180,669. Also proteases are used to increase the amount of freelyavailable nitrogen in the wort to improve fermentation.

Apart from starch other polysaccharides are present in cereal grains asfor example β-glucans (Henry, R. J. et al. J. Sci. Food Agric. 36,1243-1253, 1985). The β-glucanases present in malt are not sufficientlythermostable to be active during the brewing process. These β-glucansare highly viscous and give wort and beer filtration problems. This isthe reason why microbial β-glucanases are widely used in the brewingprocess.

Non-starch polysaccharides also include pentosans, the structure ofwhich has been widely studied recently (Gruppen, H. et al. Carbohydr.Res. 233, 45-64, 1992), in particular those of barley and malt (Vietor,R. J. et al. Carbohydr. Res. 254, 245-255, 1994). A pentosanase fromPenicillium emersonii has been said to improve the production andextraction of fermentable sugars in brewing (GB 2,150,933).

The use of xylanase B to improve wort quality has also been mentioned inWO94/14965.

Despite the advance which has been made in this area, there is still aneed for methods of beer brewing with enzyme preparations for usetherein.

SUMMARY OF THE INVENTION

The present invention discloses a process for the production ofalcoholic beverages, such as beer, to which a mixture of enzymes isadded, which mixture comprises at least an endo-β(1,4)-xylanase, anarabinofuranosidase, an alpha-amylase, an endo-protease and aβ-(1,3;1,4)-glucanase, optionally also containing a saccharifyingamylase and/or an exo-peptidase. Preferably the enzymes that arenecessary for the beer production process are provided by transgenicseeds.

The present invention further discloses transgenic seeds expressing theenzymes necessary in the beer production process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a brewing diagram showing conditions used in brewing trialsinvolving test brews prepared according to the invention and a controlbrew (see Example 10 below); and

FIG. 2 is a brewing diagram showing conditions used in brewing trialsinvolving a test brew prepared according to the invention and a controlbrew (see Examples 11 and 12, below).

DESCRIPTION OF THE INVENTION

We have now surprisingly found that the brewing process can be performedin the presence of the mixture of enzymes as claimed, with a minimalamount of malt. This process has been performed to manufacture aclassical malt beer, but it can equally well be performed in any processwhere malt is used to provide enzyme activities.

The enzymes to be used are selected from the group of enzymes which arenecessary in the brewing process. They include enzymes which areselected from the group of amylolytic enzymes, from the group ofcellulolytic enzymes, from the group of hemicellulolytic enzymes andfrom the group of proteolytic enzymes.

Amylolytic enzymes comprise enzymes like alpha-amylase, saccharifyingamylase, amyloglucosidase, exo-amylase, pullulanase.

Cellulolytic enzymes comprise enzymes like β-1,4-endoglucanase,cellobiohydrolase, β-glucosidase.

Hemicellulolytic enzymes comprise enzymes like β-1,3-1,4-glucanase,xylanase, endo-arabinanase, arabinofuranosidase, arabinoxylanase,arabinogalactanase, ferulic acid esterase.

Proteolytic enzymes comprise enzymes like exopeptidases andendopeptidases (also called prote(in)ases).

In this respect, the choice for a specific amylolytic, cellulolytic,hemicellulolytic or proteolytic enzyme is not critical for the presentinvention, besides that the choice of the enzyme should be such that theproperties of the enzyme (such as pH and temperature range) arecompatible with the specific circumstances in the brewing process.

Numerous genes encoding amylolytic, cellulolytic, hemicellulolytic andproteolytic enzymes are available to the skilled person. The genesencoding the enzymes of interest can be obtainable from any source,plant, animal or microbial. Preferably, the genes are obtainable from amicrobial source.

The endo-β-1,4)-xylanase can be obtained from a culture of Aspergillusniger (EP 0 463 706). Arabinofuranosidase is available as isoenzyme A orisoenzyme B (EP 0 506 190) or arabinoxylanhydrolase (EP 0 730 653) froma culture of Aspergillus niger. The thermostable amylase is derivablefrom Bacillus licheniformis (WO91/14772, WO92/05259), and is, forexample, commercially available under the tradename Brewers AmyliqThermostable (B.A.T.S.). Activity of the thermostable amylase isexpressed in TAU units. The endoprotease can be derived from Bacillusamyloliquefaciens, and is also commercially available under thetradename Brewers protease (+) and its activity is expressed in PCunits. From the same bacterium also the β-(1,3;1,4)-glucanase can bederived (Hofemeister et al., Gene 49, 177, 1986), which is alsocommercially available under the tradename Filtrase L 3000 (+). Activityof the glucanased is expressed in BGLU units. The optional saccharifyingamylase can also be obtained from commercially available sources(amylase from Aspergillus oryzae under the tradename Brewers Fermex forwhich the activity is expressed in FAU units), but it can also beobtained from a pure culture of Penicillium emersonii (available at theATCC (American Type Culture Collection) under number ATCC16479). Theoptional exopeptidase can be derived from a pure culture of Aspergillussojae, as has been deposited at the Centraal Bureau for Schimmelcultures(CBS), Oosterstraat 1, Baarn, The Netherlands, under number CBS 209.96(A. sojae (DS 8351) at Feb. 12, 1996).

The enzyme activities which are currently known to be necessary for beerproduction are alpha- and β-amylase to convert the starch of theendosperm to fermentable sugars, protease to degrade protein intosoluble nitrogen compounds which function as yeast nutrients, andβ-glucanase and xylanase to hydrolyse barley β-1,3-1,4-glucans andxylans, respectively, to oligosaccharides which results in a reductionof viscosity and an improvement of filterability. Thus, to provide allnecessary enzymes from an exogenous source, i.e. a microbial source,addition of at least 5 different enzymes would be required. Although onemicro-organism may be used for the production of all enzymes, differentfermentation conditions will be required for optimal production of allenzymes.

By using enzymes from microbial sources, some of the disadvantages ofthe malting process which have been mentioned before, can be overcome.However, the use of microorganisms as a source of enzymes also has itsdisadvantages:

a series of different fermentations of at least one microorganism isrequired to obtain each enzyme in a sufficient amount,

the enzyme preparations may contain undesirable side activities,

consumers do not favour additions other than plant material, water andyeast,

limited stability of an enzyme preparation during storage at roomtemperature.

The general use of transgenic seeds containing an enhanced amount ofenzyme in industrial enzyme-catalyzed processes is described ininternational patent application WO91/14772. The direct use of theenzyme-containing seed in an industrial-process circumvents the need forfirst extracting and/or isolating the enzyme. The seed can function as astable and manageable storage form of enzymes.

For one single enzyme, β-1,3-1,4-glucanase, the expression in barleyseeds has been mentioned as an alternative for exogenous addition.

East-German patent application DD 275704 discloses the construction ofan expression vector to enable the seed-specific expression of aBacillus β-1,3-1,4-glucanase in barley. However, at that time effectivetransformation of barley was not yet known. Using the seeds expressingthe Bacillus β-glucanase, a higher amount of grain can substitute formalt without obtaining serious filtration problems. However, otherenzyme activities which are necessary in the beer brewing process stillhave to be obtained from malt or supplemented exogenously.

Mannonen et al. (1993) suggest the incorporation of a fungalβ-1,3-1,4-glucanase in barley seeds. In this way, the brewing processwould be improved by the expression in the seed of a β-1,3-1,4-glucanasewhich has a higher thermostability than the endogenous barley enzyme. Inthis case, however, the intention is to take the seed through the normalmalting process. Moreover, also in this case other enzyme activitieswhich are necessary in the beer brewing process still have to beobtained from malt or supplemented exogenously.

In a preferred process of the present invention, the enzymes which arenecessary in the brewing process are expressed in transgenic seeds. Thethus-produced transgenic seeds expressing said necessary enzymes are inturn used in the beer brewing process. In this way, the use of malt isreduced, whereas the addition of exogenous microbial enzymes iscircumvented.

The transgenic seeds expressing the enzymes which are necessary in thebeer brewing process are covered by the general name MaltSeed.

Plant genera which are capable of producing the enzyme of interest intheir seeds include the species of which the grains or products fromgrains have a history of use in beer brewing. However, also plantspecies which are not commonly used for beer brewing may be used as asource of transgenic seed expressing an enzyme of interest, especiallyin those cases that only a minor amount of said transgenic seed is addedto the brewing process. Plant genera which qualify these criteria are,for instance, barley, corn, rice, wheat, sorghum, millet, oats, cassavaand the like.

The gene encoding an enzyme of interest is expressed in the plant seedusing regulatory sequences functional in a plant or plant seed. Thoseregulatory sequences include promoter sequences, terminator sequencesand, optionally, transcription enhancer sequences.

Promoter sequences may be used which lead to constitutive expression ofthe gene in the whole plant. Otherwise, promoter sequences may be usedwhich are active in directing expression of the gene to the plant seed.

Furthermore, the expression of an enzyme of interest can be directedeither to a specific cellular compartment, such as cytosol,endoplasmatic reticulum, vacuole, protein body, or to the extracellularspace, using specific targeting sequences.

The choice for a specific cellular compartment or for the extracellularspace depends on the properties of the enzyme of interest and should bemade in such a way that an optimal environment for the enzyme iscreated. For instance, the enzyme of interest should be in anenvironment that allows optimal stability of the protein during seedmaturation. In addition, the enzyme of interest should be in anenvironment where expression of the enzyme does not inhibit essentialplant metabolic processes or lead to a deleterious effect on the plantor seed viability.

In order to be capable of being expressed in a plant cell a DNA sequencecoding for the enzyme of choice will usually be provided with regulatoryelements enabling it to be recognised by the biochemical machinery ofthe host and allowing for the open reading frame to be transcribed andtranslated in the host. It will usually comprise a transcriptionalinitiation region which may be suitably derived from any gene capable ofbeing expressed in the plant cell, as well as a translational initiationregion for ribosome recognition and attachment. In eukaryotic plantcells, such an expression cassette usually comprises in addition atranscriptional termination region located downstream of said openreading frame, allowing transcription to terminate and polyadenylationof the primary transcript to occur. In addition, the codon usage may beadapted to accepted codon usage of the host plant of choice. Theprinciples governing the expression of a DNA construct in a plant cellare commonly understood by those of ordinary skill in the art and theconstruction of expressible chimeric DNA constructs is now routine.

A special type of replicon is one capable of transferring itself, or apart thereof, to another host cell, such as a plant cell, therebyco-transferring the open reading frame coding for the enzyme(s)according to the invention to said plant cell. Replicons with suchcapability are herein referred to as vectors. An example of such avector is a Ti-plasmid vector which, when present in a suitable host,such as Agrobacterium tumefaciens, is capable of transferring part ofitself, the so-called T-region, to a plant cell. Different types ofTi-plasmid vectors (vide: EP 0 116 718 B1) are now routinely being usedto transfer chimeric DNA sequences into plant cells, or protoplasts,from which new plants may be generated which stably incorporate saidchimeric DNA in their genomes. A particularly preferred form ofTi-plasmid vectors are the so-called binary vectors as claimed in (EP 0120 516 B1 and U.S. Pat. No. 4,940,838). Other suitable vectors, whichmay be used to introduce DNA according to the invention into a planthost, may be selected from the viral vectors, e.g. non-integrative plantviral vectors, such as derivable from the double stranded plant viruses(e.g. CaMV) and single stranded viruses, gemini viruses and the like.The use of such vectors may be advantageous, particularly when it isdifficult to stably transform the plant host as is for instance the casewith woody species, especially trees and vines.

The expression “host cells incorporating a chimeric DNA sequenceaccording to the invention in their genome” shall mean to comprisecells, as well as multicellular organisms comprising such cells, oressentially consisting of such cells, which stably incorporate saidchimeric DNA into their genome thereby maintaining the chimeric DNA, andpreferably transmitting a copy of such chimeric DNA to progeny cells, beit through mitosis or meiosis. According to a preferred embodiment ofthe invention plants are provided, which essentially consist of cellswhich incorporate one or more copies of said chimeric DNA into theirgenome, and which are capable of transmitting a copy or copies to theirprogeny, preferably in a Mendelian fashion. By virtue of thetranscription and translation of the chimeric DNA according to theinvention those cells will produce the enzymes. Although the principleswhich govern transcription of DNA in plant cells are not alwaysunderstood, the creation of chimeric DNA capable of being expressed isnow routine. Transcription initiation regions routinely in use forexpression of the transformed polynucleotide in a constitutive way arepromoters obtainable from the cauliflower mosaic virus, notably the 35SRNA and 19S RNA transcript promoters and the so-called T-DNA promotersof Agrobacterium tumefaciens. In particular to be mentioned are thenopaline synthase promoter, octopine synthase promoter (as disclosed inEP 0 122 791 B1) and the mannopine synthase promoter. In addition plantpromoters may be used, which may be substantially constitutive, such asthe rice actin gene promoter. For seed-specific expression, a gene ofcDNA of interest can be inserted behind promoters originating from geneswhich are specifically expressed in the plant seed. These promotersinclude promoters of genes encoding seed storage proteins, such as theBrassica napus cruciferin promoter, the Phaseolus vulgaris phaseolinpromoter, the glutelin promoter from Oryza sativa, the zein promoter ofZea mays and the hordein promoter of Hordeum vulgare.

It is further known that duplication of certain elements, so-calledenhancers, may considerably enhance the expression level of the DNAunder its regime (vide for instance: Kay R. et al. (1987), Science 236,1299-1302: the duplication of the sequence between −343 and −90 of theCaMV 35S promoter increases the activity of that promoter). In additionto the constitutive 35S promoter, singly or doubly enhanced, examples ofhigh-level promoters are the light-inducible ribulose bisphosphatecarboxylase small subunit (rbcSSU) promoter and the chlorophyll a/bbinding protein (Cab) promoter. Also envisaged by the present inventionare hybrid promoters, which comprise elements of different promoterregions physically linked. A well known example thereof is the so-calledCaMV enhanced mannopine synthase promoter (U.S. Pat. No. 5,106,739),which comprises elements of the mannopine synthase promoter linked tothe CaMV enhancer.

Specifically with monocot transformation the use of introns betweenpromoter and selectable marker gene enhances expression.

The term “promoter” thus refers to a region of DNA upstream from thestructural gene and involved in recognition and binding RNA polymeraseand other proteins to initiate transcription. A “plant promoter” is apromoter capable of initiating transcription in plant cells. A“constitutive promoter” is a promoter which is active under mostenvironmental conditions and states of development or celldifferentiation.

As regards the necessity of a transcriptional terminator region, it isgenerally believed that such a region enhances the reliability as wellas the efficiency of transcription in plant cells. Use thereof istherefore strongly preferred in the context of the present invention.

Although some of the embodiments of the invention may not be practicableat present, e.g. because some plant species are as yet recalcitrant togenetic transformation, the practicing of the invention in such plantspecies is merely a matter of time and not a matter of principle,because the amenability to genetic transformation as such is of norelevance to the underlying embodiment of the invention.

Transformation of plant species is now routine for an impressive numberof plant species, including both the Dicotyledoneae as well as theMonocotyledoneae. In principle any transformation method may be used tointroduce chimeric DNA according to the invention into a suitableancestor cell. Methods may suitably be selected from thecalcium/polyethylene glycol method for protoplasts (Krens, F. A. et al.,1982, Nature 296, 72-74; Negrutiu I. et al, June 1987, Plant Mol. Biol.8, 363-373), electroporation of protoplasts (Shillito R. D. et al., 1985Bio/Technol. 3, 1099-1102), microinjection into plant material (CrosswayA. et al., 1986, Mol: Gen. Genet. 202, 179-185), (DNA or RNA-coated)particle bombardment of various plant material (Klein T. M. et al. 1987,Nature 327, 70), infection with (non-integrative) viruses, in plantaAgrobacterium tumefaciens mediated gene transfer by infiltration ofadult plants or transformation of mature pollen or microspores (EP 0 301316) and the like. A preferred method according to the inventioncomprises Agrobacterium-mediated DNA transfer. Especially preferred isthe use of the so-called binary vector technology as disclosed in EP A120 516 and U.S. Pat. No. 4,940,838).

Although considered somewhat more recalcitrant towards genetictransformation, monocotyledonous plants are amenable to transformationand fertile transgenic plants can be regenerated from transformed cellsor embryos, or other plant material. Presently, preferred methods fortransformation of monocots are microprojectile bombardment of embryos,explants or suspension cells, and direct DNA uptake or (tissue)electroporation (Shimamoto, et al, 1989, Nature 338, 274-276).Transgenic maize plants have been obtained by introducing theStreptomyces hygroscopicus bar-gene, which encodes phosphinothricinacetyltransferase (an enzyme which inactivates the herbicidephosphinothricin), into embryogenic cells of a maize suspension cultureby microprojectile bombardment (Gordon-Kamm, 1990, Plant Cell, 2,603-618). The introduction of genetic material into aleurone protoplastsof other monocot crops such as wheat and barley has been reported (Lee,1989, Plant Mol. Biol. 13, 21-30). Wheat plants have been regeneratedfrom embryogenic suspension culture by selecting embryogenic callus forthe establishment of the embryogenic suspension cultures (Vasil, 1990Bio/Technol. 8, 429-434). The combination with transformation systemsfor these crops enables the application of the present invention tomonocots.

Monocotyledonous plants, including commercially important crops such asrice and corn are also amenable to DNA transfer by Agrobacterium strains(vide WO 94/00977; EP 0 159 418 B1; Gould J, Michael D, Hasegawa O,Ulian E C, Peterson G, Smith R H, (1991) Plant. Physiol. 95, 426-434).

For barley a preferred transformation method has been described inTingay, S. et al. (The Plant J. 11(6),. 1369-1376, 1997).

To obtain transgenic plants capable of constitutively expressing morethan one chimeric gene, a number of alternatives are available includingthe following:

A. The use of DNA, e.g. a T-DNA on a binary plasmid, with a number ofmodified genes physically coupled to a second selectable marker gene.The advantage of this method is that the chimeric genes are physicallycoupled and therefore migrate as a single Mendelian locus.

B. Cross-pollination of transgenic plants each already capable ofexpressing one or more chimeric genes, preferably coupled to aselectable marker gene, with pollen from a transgenic plant whichcontains one or more chimeric genes coupled to another selectablemarker. Afterwards the seed, which is obtained by this crossing, maybeselected on the basis of the presence of the two selectable markers, oron the basis of the presence of the chimeric genes themselves. Theplants obtained from the selected seeds can afterwards be used forfurther crossing. In principle the chimeric genes are not on a singlelocus and the genes may therefore segregate as independent loci.

C. The use of a number of a plurality chimeric DNA molecules, e.g.plasmids, each having one or more chimeric genes and a selectablemarker. If the frequency of co-transformation is high, then selection onthe basis of only one marker is sufficient. In other cases, theselection on the basis of more than one marker is preferred.

D. Consecutive transformation of transgenic plants already containing afirst, second, (etc.), chimeric gene with new chimeric DNA, optionallycomprising a selectable marker gene. As in method B, the chimeric genesare in principle not on a single locus and the chimeric genes maytherefore segregate as independent loci.

E. Combinations of the above mentioned strategies.

The actual strategy may depend on several considerations such as thepurpose of the parental lines (direct growing, use in a breedingprogramme, use to produce hybrids) but is not critical with respect tothe described invention.

It is known that practically all plants can be regenerated from culturedcells or tissues. The means for regeneration vary from species tospecies of plants, but generally a suspension of transformed protoplastsor a petri plate containing transformed explants is first provided.Shoots may be induced directly, or indirectly from callus viaorganogenesis or embryogenesis and subsequently rooted. Next to theselectable marker, the culture media will generally contain variousamino acids and hormones, such as auxins and cytokinins. It is alsoadvantageous to add glutamic acid and proline to the medium. Efficientregeneration will depend on the medium, on the genotype and on thehistory of the culture. If these three variables are controlledregeneration is usually reproducable and repeatable.

After stable incorporation of the transformed gene sequences into thetransgenic plants, the traits conferred by them can be transferred toother plants by sexual crossing. Any of a number of standard breedingtechniques can be used, depending upon the species to be crossed.

In one embodiment of the present invention, transgenic seeds are usedwhich are engineered in such a way that these produce one individualenzyme, allowing for the flexible production of enzyme mixtures withevery enzyme activity ratio which is desired. In another embodiment,more than one enzyme activity may be contained in the seeds of anindividual transgenic plant line.

The transgenic seeds containing the enzymes of interest can be addedtogether at a desired stage of the brewing process. Alternatively,transgenic seeds containing an enzyme of interest can be addedindividually, each at a desired point of the process.

The process of the present invention enables the development of a maltwith a high level of flavour and colour, without having to deal with theenzyme activity of the malt. In the process of the present invention theuse of malt is largely bypassed. Only a minor amount of malt may stillbe necessary to provide the beer with flavour and colour.

The transgenic seeds containing the desired enzymes can be applied inthe most optimal ratio with the grain and, optionally, with a sufficientamount of malt for flavour and colour. The transgenic seeds can be mixedbeforehand with the grain and the malt. Alternatively, each compound,grain, transgenic seed and malt, can be added at separate stages of thebeer brewing process.

Preferably, the individual compounds, transgenic seeds, grain and malt,or the mixture of transgenic seed, grain and malt are milled beforeaddition to the brewing process.

The transgenic seed contains the desired enzymes at an average levelthat ranges from 0.001-2.5%, preferably from 0.01-1.0%, more preferablyfrom 0.05-0.25% by weight of the seed. Depending on the level ofexpression in the seed only part or the total of the seeds normally usedin the brewing process can be replaced by transgenic seeds. When highlevels of expression are reached it is also possible to add thetransgenic seeds of other plant genera not normally used in the brewingprocess without changing too much to the brewing mix.

In the case of malt, preferably part of the malt has received a specialtreatment, as compared to traditional malt, wherein the malt has beenheated during kilning to maximize production of colour and flavourcompounds. The remaining enzyme activity is negligible after thistreatment.

Expression of enzymes in seed as is disclosed in the present inventionprovides the possibility to circumvent the addition of exogenousmicrobial enzymes to the brewing process. The costs of the production oftransgenic seeds, containing the enzymes are much lower than the costsof the production of enzymes by fermentative processes. Furthermore,transgenic seeds are more conveniently used, since they provide a stableand manageable storage form of enzymes and are easy to handle. The costreduction and the convenience of use which are coupled with the use oftransgenic seeds are especially relevant in the process of the presentinvention, because the need is circumvented to apply several differentenzymes from several microbial fermentations in the beer brewingprocess.

The course of the process of the present invention is much morepredictable than the course of a process using malt as a source ofenzymes, since the transgenic seeds contain no other enzyme activitiesat high levels than those expressed by way of the introduced genes. Inaddition, the course of the process of the present invention is muchmore predictable than the course of a process using microbial enzymes,since microbial enzyme preparations display undefined and varying levelsof side activities.

One of the areas in which it can be especially used is in Africancountries where malt importation is banned. The enzymes in that casecould be expressed in sorghum, which then could be added to the brewingprocess.

Next to the applications in the brewing of alcoholic beverages alsoother applications can be foreseen, in which MaltSeed can replace maltedgrains.

Malted barley and/or malted wheat are used by millers to standardize thediastasic power of flour. Also hemicellulases (such as xylanase) areadded to the flour to improve the gas retention capacity of doughs madefrom the flour. The addition or use of enzymes in flour can be replacedby using MaltSeed, which is very suitable since the enzymes can be addedin the form of grains which anyhow are used. Identically also in thebaking process enzymes can be added as a replacement for the malt whichis present in many doughs. Enzymes which are improving the bakingprocess are xylanase,amylase, arabinofuranosidase, exopeptidase. Alsothe glucose oxidase from Aspergillus niger can be expressed in seeds forbakery purposes.

EXAMPLE 1 Activity Measurement of Endo-β-1,4-xylanase

Endoxylanase is obtained from a pure culture of Aspergillus niger in asterile tank and medium. The culture medium contains appropriate carbonand nitrogen sources just as mineral salts. The fermentation is carriedout at a constant temperature between 30-40° C. and pH is maintainedwithin the range 3-5.

The activity of the enzyme is measured by hydrolysis of xylan from oatspelts suspended (35 g/l) in 1M glycine buffer pH 2.75. The viscosity ofthis solution is determined by using a capillary viscometer (Ubbelhodetype) at 47° C. The time dt needed for the upper meniscus of the liquidto fall down between two reference points is measured within time T. Theslope of the plot T versus 1/dt yields an apparent kinetic constant. 1Lyx unit is the amount of enzyme needed to reach a value of 1 min-1 forthat kinetic constant.

EXAMPLE 2 Activity Measurement of Exopeptidase

A production strain of Aspergillus sojae (DS 8351) is cultured.Exopeptidase activity is expressed as Leucine aminopeptidase units(Leu-A): 1 Leu-A is the amount of enzyme needed to produce 1 μmolp-nitroaniline per minute at pH 7.2 and 20° C. fromL-leucine-p-nitroaniline. The test is performed as follows:

Leucine paranitroanilid (SIGMA) is dissolved in water at a concentrationof 9 mM. 1 ml of the solution is mixed with 1.5 ml 0.1 M phosphatebuffer pH 7.2. At t=0, 0.5 ml enzyme is introduced and left for reactionat 20° C. Fifteen minutes later, 1 ml 1N HCl is added to stop thereaction. A blank is run with 1N HCl being introduced at t=0. Opticaldensity is determined for the blank (OD_(blank)) and for the assay(ODassay) at 400 nm. Activity is calculated as follows:$A = {{\frac{\left( {{OD}_{blank} - {OD}_{assay}} \right)}{9.8 \times 15} \times \frac{4}{0.5}\quad {Leu}} - {A\text{/}{ml}}}$

EXAMPLE 3 Activity Measurement of Arabinofuranosidase

Isoenzyme A or isoenzyme B or arabinoxylanhydrolase have been obtainedfrom a culture of Aspergillus niger or Aspergillus nidulans strains.Activity of isoenzymes A and B is measured by the hydrolysis ofp-nitrophenyl-alpha-L-arabinofuranoside. 1 ARF unit is the amount ofenzyme needed to liberate 1 μmol p-nitrophenol per minute under the testconditions described in Gunata Z. et al. (J. Agric. Food Chem. 38, 772,1989).

EXAMPLE 4 Activity Measurement of Saccharifying Amylase

Saccharifying amylase is obtained from a pure culture of Penicilliumemersonii in a sterile tank and medium, which contains appropriatecarbon and nitrogen source just as mineral salts. The tank is fed withmaltodextrines 10-30 hours (preferably 24 h) after the start of thefermentation. Temperature is maintained in the range of 40-50° C.(preferably 45° C.) and pH is maintained in the range 4.5-5.5(preferably 5.0). The fermentation is stopped 40-55 h (preferably 48 h)after start.

Saccharifying amylase activity is measured according to the BETAMYLtest, commercially available from MEGAZYME, Ireland. 1 BTU is the amountof enzyme needed to produce 1 μmol p-nitrophenol at pH 6.2 and 40° C.from Megazyme's commercial substrate.

EXAMPLE 5 Preparation of Wort using Microbial Enzymes

A wort was prepared from crude barley grains, variety PLAISANT. Barleywas ground with the EBC MIAG mill in order to reach filter press typegranulometry. 57 g of the obtained milled barley was suspended in 300 mlwarm water (50° C.) and containing:

650 Lyx units endo-β-(1,4)-xylanase

850 ARF units arabinofuranosidase

18 mg B.A.T.S. (thermostable amylase)

6 mg Brewers Protease (+) (endo-protease)

1 mg Filtrase L3000 (+) (β-(1,3;1,4)-glucanase)

The temperature was maintained at 50° C. for 30 minutes and then raisedup to 63° C. (rate 1° C./min); the temperature was further maintained at63° C. for 30 minutes and then raised up to 72° C. (rate 1° C./min) andmaintained at that temperature for 30 minutes. It was finally heated upto 76° C. (rate 1° C./min) and maintained at that temperature for 5minutes. Water was added to compensate for water evaporation. The mashwas then poured into a funnel containing Schleicher and Schull paperfilter. From the density of the filtered wort, yield was determined asdone in any brewery; also viscosity and free amino acids (FAA) levelswere determined according to standard EBC procedures. The yield was71.5%, viscosity was 2.52 mPa.s and 66 mg/l of free amino acids weremeasured.

EXAMPLE 6 Comparison of Saccharifying Amylases

A wort was prepared from crude barley grains, variety PLAISANT. Barleywas ground with the EBC MIAG mill in order to reach filter press typegranulometry. 57 g of the obtained milled barley was suspended in 300 mlwarm water (50° C.) and containing:

650 Lyx units endo-β-(1,4)-xylanase

850 ARF units arabinofuranosidase

18 mg B.A.T.S. (thermostable amylase)

6 mg Brewers Protease (+) (endo-protease)

100 Leu-A units exopeptidase

1 mg Filtrase L3000 (+) (β-(1,3;1,4)-glucanase)

According to Table 1 saccharifying enzymes were added to the brewmixture.

TABLE 1 Doses of saccharifying enzymes Brew no. Saccharifying enzyme 1none  0 2 Brewers Fermex 510 FAU 3 Amylase from P. emersonii  10 BTU 4Brewers Fermex + amylase 510 FAU + 10 BTU from P. emersonii

The temperature was maintained at 50° C. for 30 minutes and then raisedup to 63° C. (rate 1° C./min); the temperature was further maintained at63° C. for 30 minutes and then raised up to 72° C. (rate 1° C./min) andmaintained at that temperature for 30 minutes. It was finally heated upto 76° C. (rate 1° C./min) and maintained at that temperature for 5minutes. Water was added to compensate for water evaporation. The mashwas then poured into a funnel containing Schleicher and Schull paperfilter. From the density of the filtered wort, yield was determined asdone in any brewery; also viscosity and free amino acids (FAA) levelswere determined according to standard EBC procedures.

Results of the measured yield, viscosity and FAA given in Table 2 showthe effects of the saccharifying enzyme of Penicillium emersonii as asubstitute of Brewers Fermex whereas no real synergism can be expectedfrom the use of both enzymes. Particularly surprising is the quitepositive effect of the amylase of Penicillium emersonii on FAA increaseand viscosity reduction.

TABLE 2 Results Viscosity FAA (12 Brew no. Yield (%) (mPa.s) Plato)(mg/l) 1 71.2 3.12 116 2 74.6 2.73 114 3 78.2 1.99 153 4 79.6 1.99 152

EXAMPLE 7 Construction of a Binary Vector Containing a Seed-specificExpression Cassette

An expression construct is constructed in such a way that seed-specificexpression is obtained, using sequences of Oryza sativa L. glutelinstorage protein (Zheng et al., Plant Physiol (1995) 109; 77-786). Thesesequences may be replaced by those from similar seed-specific genes toachieve the same goal as is the objective of this invention.

For the construction of the expression construct for seed-specificexpression, the promoter and terminator sequences from the glutelin(Gtl) gene of Oryza saliva L. are synthesized using PCR technology withthe genomic clone Gtl (Okita at al, J. Biol. Chem. 264, 12573-12581,1989) as a template. This gene shows see-specific expression and itscoding and flanking sequences have been determined (EMBL. GenbackNucleotide Sequence Database accession number D00584)). Two sets ofoligonucleotides arc synthesized. One to allow amplification of a 2.4 kbfragment containing the Gtl 5′ flanking region encoding as an XhoI/Sphlfragment:

5′Gt1.1 5′ GCACAATTCTCGAGGAGACCG 3′ (SEQ ID NO:6) 5′Gt1.25′ ATGGATGGCATGCTGTTGTAG 3′ (SEQ ID NO:5)

The other amplification of the 3′ flanking sequences as a BamHI/EcoRIfragment (725 bp):

3′Gt1.3 5′ CCTCTTAAGGATCCAATGCGG 3′ (SEQ ID NO:7) 3′Gt1.45′ CTTATCTGAATTCGGAAGCTC 3′ (SEQ ID NO:8)

The oligos are designed to contain suitable restriction sites at theirtermini to allow direct assembly of the expression construct afterdigestion of the fragments with restriction enzymes. Genes for theenzymes in the mixture according to the invention can be obtained fromliterature for the endo-xylanase (Mol. Microbiol. 12, 479-490, 1994),for the arabinofuranosidase isoenzyme A and isoenzyme B (EP 0 506 190),for the amylase from Bacillus licheniformis (EP 0 449 376), for theprotease from Bacillus amyloliquefaciens (J. Bact. 159, 811-819) and forthe glucanase form Bacillus amyloliquefaciens (Gene 49, 177-187, 1986).The genes for the saccharifying amylase from Penicillium and for theexopeptidase from Aspergillus sojae can easily be elucidated for aperson skilled in the art from the pure enzyme obtainable from thecultures indicated in the description.

The codon usage of the genes encoding the enzymes to be expressed inseeds is optimized for expression in monocot seeds. In order to do thisthe complete gene is made synthetically, a BspHI site is introduced atthe ATG start codon and a BamHI site is introduced down-stream of theTAA stop codon both for cloning purposes.

The 2.4 kb PCR product containing the 5′ flanking region of Gtl isdigested with XhoI/SphI and cloned in a vector pSL1180 linearized withXhoI/SphI. The resulting vector is linearized with SphI/BamHI and usedas a vector in a three-way ligation with the synthetic enzyme-encodinggene and, optionally an oligonucleotide duplex coding for a targetingsignal. Targeting can be effectuated to the vacuole, to the apoplast, tothe amyloplast or (with e.g. a KDEL-retention signal) to theendoplasmatic reticulum.

From this vector a fragment is isolated, containing the fusions of theGtl glutelin promoter, optional signal sequence and the synthetic gene.This fragment is cloned in a three-way ligation with the 725 bp PCRproduct containing the 3′ terminator sequence of Gtl digested withBamHI/EcoRI into binary vector pMOG22 (in E. coli K-12 strain DH5-alpha,deposited at the Centraal Bureau voor Schimmelcultures on Jan. 29, 1990under accession number CBS 101.90).

EXAMPLE 8 Construction of a Binary Vector Containing the Endo-xylanaseGene in the Seed-specific Expressing Cassette

The endoxylanase gene from Aspergillus niger is used to optimise forcodon usage in barley. The resulting DNA sequence is depicted in SEQ IDNO:1.

For the expression of the endoxylanase gene extracellular targeting isaccomplished by the oligonucleotide duplex

PRS 1 (SEQ ID NO:3)5′       AACTTCCTCAAGAGCTTCCCCTTTTATGCCTTCCTTTGTTTTGGCCAATACTTTGTAGCTGTTACGCATGC    3′PRS 2 (SEQ ID NO:4)3′ GTACTTGAAGGAGTTCTCGAAGGGGAAAATACGGAAGGAAACAAAACCGGTTATGAAACATCGACAATGCGTACGGTACC5′

encoding the signal peptide of the tobacco PR-S protein and for thethree-way ligation the synthetic xylanase gene digested withBspGHi/BamHI is used.

From this vector a 3.1 Kb XhoI/BamHI fragment is isolated, containingthe fusions of the Gtl glutelin promoter, PR-S signal sequence and thesynthetic xylanase gene. This fragment is cloned in a three-way ligationwith the 725 bp PCR product containing the 3′ terminator sequence of Gtldigested with BamHI/EcoRI into binary vector pMOG22. The resultingvector is designated pMOG1265.

EXAMPLE 9 Barley Transformation

The method used for transformation of immature embryos of Hordeumvulgare cv. Golden Promise using Agrobacterium tumefaciens is generallyas described in Tingay, S. et al., The Plant J. 11(6), 1369-1376, 1997.In short, the protocol is as follows:

Donor plants for starting material are grown in a phytotron at 10-20° C.with a 16 hr light period at 10,000-30,000 lux and 50-95% RH. Immatureseed are harvested 10-15 days after pollination and sterilized in ableach solution for 20-40 minutes. Immature embryos are excised from theyoung caryopses and the embryonic axis is removed with a scalpel blade.The explants are placed scutellum-side up on callus induction medium andincubated at 24° C. in the dark for a period ranging from 16 hours till7 days.

Embryos are immersed in an Agrobacterium suspension, approximately0.1-10×10⁹ bacteria per ml, in which the Agrobacterium contains theconstructs comprising the DNA encoding for the enzyme of choice, for 5to 20 minutes and then transferred to callus induction medium.Thereupon, embryos are incubated for 2 or 3 days at 24° C. in the dark.After coculture, embryos are transferred to callus induction mediumcontaining antibiotics to kill Agrobacteria, directly combined with aselective agent to start the selection process of transgenic cells. Theselection process occurs for up to 8 weeks. Resistant embryogenic calluslines are transferred to regenration medium and incubated in increasinglight intensity (500 to 3000 lux) with a 16 hr light period at 24° C.Regenerating plantlets are transferred to hormone free or high cytokinincontaining callus induction medium with or without selective agent.After development of a root system, plantlets are transferred to soiland grown to maturity with self-pollination.

EXAMPLE 10

Three complete pilot brewing trials were performed. Two test brews weredone with a severely reduced amount of malt (20% of the raw material)and the control with a normal amount of malt (See Table 3). The two testbrews differed in the milling and filtering technology (See Table 3).The brewing diagram for all brews is shown in FIG. 1.

TABLE 3 Raw material composition, filtering and milling Control Testbrew 1 Test brew 2 Pils malt 75% 20% 20% Maize grits 25% 25% 25%Unmalted barley — 55% 55% Filter Lautertun Lautertun Meura 20001 MillingCylinder Cylinder Hammer

From the control mixture 8% malt was used in the cereal cooker togetherwith the maize grits, the other 67% malt was added to the conversionvessel. For the test brews 8% unmalted barley was used in the cerealcooker together with the maize grits, the other 47% unmalted barley wasadded to the conversion vessel together with the malt.

TABLE 4 Enzymes codes and amounts added per 100 kg raw material EnzymeCereal Conversion Code Enzyme cooker vessel 1 Endo-β(1,4)-xylanase117,000 Lyx 2 Arabinofuranosidase 112,500 ARF 3 B.A.T.S. 16.5 g 4Brewers Fermex 75 g 5 Brewers protease (+) 75 g 6 Exo-peptidase — 7Filtrase L3000 (+) 23 g

The enzymes 1, 2, 4, 5 and 7 were added to the conversion vessel, whileenzyme 3 was added to the cereal cooker (see Table 4 for enzyme codesand amounts added).

Wort processing results (mashing and lautering) were similar for thetest brews and the control. The two test brews did perform similarly inthe brewhouse. The test brews gave roughly the same wort, showing thatthe differences in filtering and milling were not essential. From ataste comparison of the test and control beers it was concluded that allthree beers had a quite similar profile. A stronger mouthfeel wasobserved for the test brews in comparison with the control. This may bedue to a higher dextrin level that was found in the analysis of thewort. The amino acid levels in the wort were, although acceptable, lowerin the test brews in comparison with the control. The amino acid levelscan be increased by addition of the exopeptidase (see example 11). Thetest brew beers were classified by the tasting panel as good pilsenerbeers, showing that the partial replacement of malt by the enzymemixture resulted in a beer that is comparable to a beer manufacturedwith an amount of malt that is commonly used in the brewing industry.

EXAMPLE 11

Two complete pilot brewing trials were performed. The test brew was donewith a reduced amount of malt and the control with a normal amount ofmalt (See Table 5). Filtering was done on the Lautertun. For the brewingdiagrams of test brew 1 and the control see FIG. 2.

TABLE 5 Raw material composition Control Test brew 1 Pils malt 75% 20%Maize grits 25% 25% Unmalted barley — 55%

From the control mixture 8% malt was used in the cereal cooker togetherwith the maize grits, the other 67% malt was added to the conversionvessel. For the test brew 8% unmalted barley was used in the cerealcooker together with the maize grits, the other 47% unmalted barley wasadded to the conversion vessel together with the malt.

In the test brew, enzymes 1-7 were added to the conversion vessel, whileenzyme 3 was added to the contents of the cereal cooker as well (seeTable 6 for enzyme codes and amounts of enzyme used.

Wort processing results (mashing and lautering) were similar for thetest brews and the control. The free amino acid nitrogen content in thewort was similar for the test brew and the control. The test brewresulted in beer that by tasting as considered to have a quite similarprofile as the control. The test brew beer classified by the tastingpanel as good pilsener beer, showing that the partial replacement ofmalt by the enzyme mixture resulted in a beer that is comparable to abeer manufactured with an amount of malt that is common use in thebrewing industry.

TABLE 6 Enzymes codes and amounts added per 100 kg raw material in thetest brew Enzyme Conversion Code Enzyme Cereal cooker vessel 1Endo-β(1,4)-xylanase 117,000 Lyx 2 Arabinofuranosidase 112,500 AFR 3B.A.T.S. 16.5 g 33.5 g 4 Brewers Fermex   75 g 5 Brewers protease (+)  75 g 6 Exo-peptidase 105,000 LeuA 7 Filtrase L3000 (+)   23 g

EXAMPLE 12

Transgenic barley grains from seven lines, each expressing one of theenzymes shown in Table 9 are mixed in such amounts that the resultingMaltSeed, when mixed in as 10% of the crude barley, provides enzymeactivities as shown in Table 9. The MaltSeed preparation is mixed andground together with the non-transgenic barley, together constituting55% of the raw material in the test brew. The raw material compositionsin the cereal cooker and the conversion vessel for the test brew areshown in Table 8. For the control 8% malt was used in the cereal cookertogether with the maize grits. The rest of the raw material was used inthe conversion vessel. The distribution of MaltSeed gives the enzymeactivities as shown in Table 10 in the conversion vessel and the cerealcooker.

Two brewing trials were performed. The test brew was done with a reducedamount of malt and the control with a normal amount of malt (See Table7). For the test brew a higher sacharification temperature was used (SeeTable 7). Filtering was done on the Lautertun. For the brewing diagramof the control and test brew see FIG. 2.

TABLE 7 Composition raw material Control Test brew 1 Pils malt 75% 20%Maize grits 25% 25% Unmalted barley + MaltSeed — 55%

TABLE 8 Raw material composition per 100 kg total in the test brewCereal cooker Conversion vessel Raw material (kg) (kg) Malt 20 Unmaltedbarley 7.2 42.3 MaltSeed 0.8 4.7 Maize grits 25 Total (kg) 33 67

Wort processing results (mashing and lautering) were similar for thetest brew and the control. The test brew resulted in beers that bytasting was considered to have a similar profile as the control. Thetest brew was classified as a classical malt beer by tasting, showingthat malt can be (partially) replaced by enzymes provided throughtransgenic seeds expressing these.

TABLE 9 Amount of enzyme activity added through addition of MaltSeed(units per 100 kg raw material) in test brew 1 Enzyme Cereal ConversionCode Enzyme Cooker vessel 1 Endo-β(1,4)-xylanase  19,915 Lyx 117,000 Lyx2 Arabinofuranosidase  19,149 ARF 112,500 ARF 3 B.A.T.S. 107,250 TAU630,094 TAU 4 Brewers Fermex  57,872 FAU 340,000 FAU 5 Brewers protease(+) 115,745 PC 680,000 PC 6 Exo-peptidase  17,872 Leu-A 105,000 Leu-A 7Filtrase L3000 (+)  11,701 BGLU  68,745 BGLU

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 8(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 557 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO     (ix) FEATURE:           (A) NAME/KEY: CDS          (B) LOCATION: 1..555           (D) OTHER INFORMATION: #/product= “mature protein”     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #1: ATG AGC GCG GGA ATC AAC TAC GTC CAG AAC TA#C AAT GGC AAC CTC GGC  48 Met Ser Ala Gly Ile Asn Tyr Val Gln Asn Ty#r Asn Gly Asn Leu Gly   1               5  #                 10 #                 15 GAC TTT ACT TAC GAC GAG TCA GCG GGA ACT TT#C AGC ATG TAT TGG GAG  96 Asp Phe Thr Tyr Asp Glu Ser Ala Gly Thr Ph#e Ser Met Tyr Trp Glu              20      #             25     #             30 GAT GGC GTG TCC TCA GAC TTC GTC GTG GGA CT#G GGC TGG ACC ACT GGA 144 Asp Gly Val Ser Ser Asp Phe Val Val Gly Le#u Gly Trp Thr Thr Gly          35          #         40         #         45 TCA TCC AAT GCG ATC ACC TAC AGC GCC GAG TA#C TCC GCG TCA GGA TCA 192 Ser Ser Asn Ala Ile Thr Tyr Ser Ala Glu Ty#r Ser Ala Ser Gly Ser      50              #     55             #     60 GCC TCC TAT CTG GCC GTG TAC GGA TGG GTG AA#C TAC CCG CAG GCC GAG 240 Ala Ser Tyr Leu Ala Val Tyr Gly Trp Val As#n Tyr Pro Gln Ala Glu  65                  # 70                 # 75                  # 80 TAC TAC ATC GTG GAG GAT TAC GGA GAT TAC AA#C CCA TGC AGC TCA GCG 288 Tyr Tyr Ile Val Glu Asp Tyr Gly Asp Tyr As#n Pro Cys Ser Ser Ala                  85  #                 90 #                 95 ACC TCC CTC GGA ACT GTG TAC AGC GAC GGC TC#C ACC TAC CAG GTC TGC 336 Thr Ser Leu Gly Thr Val Tyr Ser Asp Gly Se#r Thr Tyr Gln Val Cys             100       #           105      #           110 ACC GAC ACC CGC ACT AAC GAG CCG TCA ATC AC#C GGC ACT TCC ACC TTC 384 Thr Asp Thr Arg Thr Asn Glu Pro Ser Ile Th#r Gly Thr Ser Thr Phe         115           #       120          #       125 ACC CAG TAC TTC AGC GTG CGC GAG TCC ACT CG#C ACC TCA GGA ACC GTG 432 Thr Gln Tyr Phe Ser Val Arg Glu Ser Thr Ar#g Thr Ser Gly Thr Val     130               #   135              #   140 ACC GTC GCG AAC CAC TTC AAC TTC TGG GCG CA#G CAC GGA TTC GGC AAC 480 Thr Val Ala Asn His Phe Asn Phe Trp Ala Gl#n His Gly Phe Gly Asn 145                 1 #50                 1#55                 1 #60 AGC GAC TTT AAC TAC CAG GTG GTC GCA GTG GA#G GCA TGG TCA GGA GCG 528 Ser Asp Phe Asn Tyr Gln Val Val Ala Val Gl#u Ala Trp Ser Gly Ala                 165   #               170  #               175 GGC TCA GCG TCC GTC ACT ATC AGC TCC TG  #                   #      557 Gly Ser Ala Ser Val Thr Ile Ser Ser            180       #           185 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 185 amino #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:Met Ser Ala Gly Ile Asn Tyr Val Gln Asn Ty #r Asn Gly Asn Leu Gly  1               5  #                 10  #                 15Asp Phe Thr Tyr Asp Glu Ser Ala Gly Thr Ph #e Ser Met Tyr Trp Glu             20      #             25      #             30Asp Gly Val Ser Ser Asp Phe Val Val Gly Le #u Gly Trp Thr Thr Gly         35          #         40          #         45Ser Ser Asn Ala Ile Thr Tyr Ser Ala Glu Ty #r Ser Ala Ser Gly Ser     50              #     55              #     60Ala Ser Tyr Leu Ala Val Tyr Gly Trp Val As #n Tyr Pro Gln Ala Glu 65                  # 70                  # 75                  # 80Tyr Tyr Ile Val Glu Asp Tyr Gly Asp Tyr As #n Pro Cys Ser Ser Ala                 85  #                 90  #                 95Thr Ser Leu Gly Thr Val Tyr Ser Asp Gly Se #r Thr Tyr Gln Val Cys            100       #           105       #           110Thr Asp Thr Arg Thr Asn Glu Pro Ser Ile Th #r Gly Thr Ser Thr Phe        115           #       120           #       125Thr Gln Tyr Phe Ser Val Arg Glu Ser Thr Ar #g Thr Ser Gly Thr Val    130               #   135               #   140Thr Val Ala Asn His Phe Asn Phe Trp Ala Gl #n His Gly Phe Gly Asn145                 1 #50                 1 #55                 1 #60Ser Asp Phe Asn Tyr Gln Val Val Ala Val Gl #u Ala Trp Ser Gly Ala                165   #               170   #               175Gly Ser Ala Ser Val Thr Ile Ser Ser             180      #           185 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 71 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA   (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:           (A) ORGANISM: Nicotiana  #tabacum    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:AACTTCCTCA AGAGCTTCCC CTTTTATGCC TTCCTTTGTT TTGGCCAATA CT#TTGTAGCT     60 GTTACGCATG C                #                  #                   #       71 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 80 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA   (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4:CCATGGCATG CGTAACAGCT ACAAAGTATT GGCCAAAACA AAGGAAGGCA TA#AAAGGGGA     60 AGCTCTTGAG GAAGTTCATG             #                  #                   # 80 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 21 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA   (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5:ATGGATGGCA TGCTGTTGTA G            #                  #                   #21 (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 21 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA   (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6:GCACAATTCT CGAGGAGACC G            #                  #                   #21 (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 21 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA   (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7:CCTCTTAAGG ATCCAATGCG G            #                  #                   #21 (2) INFORMATION FOR SEQ ID NO: 8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 21 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA   (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8:CTTATCTGAA TTCGGAAGCT C            #                  #                   #21

What is claimed is:
 1. In a process for preparing an alcoholic beveragecomprising heating, steeping or mixing a plurality of raw materials toform a brew, the improvement comprising adding to the brew during anystage of its formation a non-malt component or a plurality of non-maltcomponents collectively comprising a mixture of enzymes including atleast an endo β-(1,4)-xylanase, an arabinofuranosidase, analpha-amylase, an endo-protease and a β-(1,3-1,4)-glucanase, wherein theenzymes are present in respective amounts sufficient to provide thealcoholic beverage with a taste profile of a malt brew having a highermalt content than said alcoholic beverage, and wherein the component orplurality of components is present in an individual seed or in aplurality of seeds.
 2. A process according to claim 1, wherein theindividual seed is from a transgenic plant line, said individual seedcontaining at least one of said enzymes.
 3. A process according to claim1, wherein the plurality of the components collectively comprising saidmixture of enzymes is added to the brew, each of said plurality ofcomponents being present in the plurality of seeds with each seed in theplurality of seeds containing a respective one of said enzymes.
 4. Aprocess according to claim 3, wherein each of the plurality of seeds isfrom a different transgenic plant line, the seed from each of thedifferent transgenic plant lines containing a respective one of saidenzymes.
 5. A process according to claim 1, wherein the individual seedcomprises a plurality of the enzymes.
 6. A process according to claim 5,wherein the individual seed is from a transgenic plant line thatproduces seed containing said plurality of enzymes.
 7. A processaccording to claim 2, wherein the tranagenic plant line is a barleyplant line.
 8. A process according to claim 4, wherein each of thedifferent transgenic plant lines is a barley plant line.
 9. A processaccording to claim 6, wherein the transgenic plant line is a barleyplant line.
 10. A process according to claim 4, wherein at least one ofthe enzymes is a microbial enzyme.
 11. A process according to claim 2,wherein the at least one enzyme is a microbial enzyme.
 12. A processaccording to claim 6, wherein at least one of the plurality of enzymesis a microbial enzyme.
 13. A process according to claim 11, wherein thetransgenic plant line is a barley plant line.
 14. A process according toclaim 12, wherein the transgenic plant line is a barley plant line. 15.A process according to claim 10, wherein the transgenic plant line is abarley plant line.